Download The Protein Truncation Test

THE PROTEIN
TRUNCATION TEST
4
C H A P T E R
About the Image:
This diagram depicts
production of a truncated
protein (right) as compared
to a normal length protein
(left) using the Protein
Truncation Test. This test
has been used, in vitro, to
determine whether a gene
mutation results in a
shortened translation
product that may lead to a
cancerous cell.
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THE PROTEIN
TRUNCATION TEST
Chapter Four: The Protein Truncation Test
Contents
Page
Introduction ............................................................................................................ 19
PTT Principle .......................................................................................................... 20
Source Considerations................................................................................................ 21
Detection and Primer Design ........................................................................................ 22
Introduction
References
1. Lesieur, A. et al. (1994) Diag.
Mol. Pathol. 3, 75.
2. Lee, K-O. et al. (1996) J.
Biochem. Mol. Biol. 29, 241.
3. den Dunnen, J.T. et al. (1989)
Am. J. Hum. Genet. 45, 835.
4. Chamberlain, J.S. et al. (1988)
Nucl. Acids Res. 16, 11141.
5. Loenig, M. et al. (1987) Cell 50,
509.
6. Orita, M. et al. (1989) Genomics
5, 874.
Mutations in a gene can range from large deletions to single point mutations. Many of the large
deletions or translocations can be readily detected. For example, 95% of the cases of chronic myelogenous leukemia contain the Philadelphia chromosome, which is a translocation of part of
chromosome 22 to chromosome 9. The abnormality can be detected by Southern blotting as aberrant or additional reactive bands when compared to normal samples (1). In this translocation, the
abl proto-oncogene is translocated into the bcr gene resulting in the expression of a bcr-abl fusion
protein. The chimeric transcript can be readily detected by RT-PCR(f) (2). Point mutations or small
deletions, however, are much more difficult to detect. In Duchenne muscular dystrophy (DMD), for
example, one third of the reported mutations in the gene DMD are not detectable as intragenic deletions or duplications (3–5). Techniques such as single strand confirmation polymorphism (6) can
detect sequence differences but cannot distinguish between a polymorphism that may result in no
phenotype (e.g., conservative amino acid change) and a polymorphism with a definite effect on the
protein produced (e.g., premature termination of sequence).
A rapid solution to these problems can be achieved through a procedure known as PTT (protein
truncation test).
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PROMEGA IN VITRO RESOURCE
References (continued)
PTT Principle
7. Roest, P.A.M. et al. (1993) Hum.
Mol. Genet. 2, 1719.
8. Baklanov, M.M. et al. (1996)
Nucl. Acids Res. 24, 3659.
9. TNT ® Quick Coupled
Transcription/Translation
Systems Technical Manual
#TM045, Promega Corporation.
10. Kozak, M. (1986) Cell 44, 283.
11. pGEM ®-T and pGEM ®-T Easy
Vector Systems Technical
Manual #TM042, Promega
Corporation.
sequence at the 5′-end that directs transcription. Usually, additional nucleotides are present
upstream of the T7 promoter. Even the addition
of a single G nucleotide upstream of the promoter increases the transcriptional efficiency
(8). While T7 is the most commonly used promoter, T3 RNA polymerase promoter can be
used as well. SP6 promoters are not well-suited
for coupled transcription/translation of linear
DNA (9). Promega offers a system specifically
for the expression of PCR(f) products, the TNT®
T7 Quick for PCR DNA(c,d,e) (Cat.# L5540)*. A
3–6bp spacer separates the promoter
sequence from an optimal eukaryotic translation
initiation sequence, which includes the initiation
codon ATG. The optimal eukaryotic translation
initiation sequence is referred to as a Kozak
consensus sequence (10). The bacteriophage
promoter, spacer and Kozak sequence are followed by sequences specific to the target
(Table 2). At the 3´-end of the target, the primer
can include a stop codon if the amplified
sequence does not contain the native stop
codon (9). Restriction enzyme recognition sites
can also be engineered into both primers to aid
A simple way to judge whether a mutation
results in a truncation or not, is to translate the
protein in vitro. Roest et al. (7) developed the
protein truncation test (PTT) to rapidly screen for
these mutations. PTT is composed of four steps:
i) isolation of nucleic acid, either genomic DNA,
total RNA or poly (A)+ RNA; ii) amplification of a
specific region of the gene of interest; iii) in vitro
transcription and translation of the product of the
amplification reaction; and iv) detection of the
translation products. The shorter protein
products of the mutant alleles are easily distinguished from the full-length protein product of
the normal allele (Figure 1). PTT has been used
to analyze many genes in addition to DMD
(Table 1).
*For Laboratory Use.
Amplified sequences for PTT can be generated
across the entire protein coding sequence or
they can be generated to specific exons. The
key feature of PTT is a specifically designed
PCR primer to allow coupled in vitro transcription/translation of the amplified sequence. The
primer contains a T7 bacteriophage promoter
Cells from blood
or tissue sample
RNA
Genomic DNA
1
Exons
2
3 4
5
1
Exons
2 3 4 5
mRNA
Reverse
Transcription
cDNA
PCR
dsDNA
ATG
Forward Primer
+
Reverse Primer
T7
dsDNA
ATG
PCR
T7
in vitro
Transcription/
Translation
RNA
Agarose gel electrophoresis
of PCR products
AUG
Protein
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– Full-length protein
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– Truncated protein
Figure 1. Schematic diagram of the Protein Truncation Test.
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THE PROTEIN
TRUNCATION TEST
in subcloning of the PCR product if verification
of a mutation is needed. The advent of PCR
product cloning vectors has abrogated the
need for inclusion of the restriction sites into
PCR primers (11).
Source Considerations
The PTT test can be applied to individual exons
of a gene via amplification of genomic DNA.
Hogervorst et al. (12) analyzed genomic DNA of
stored heparinized blood for mutations in the
breast and ovarian cancer gene, BRCA1.
Greater than 75% of the reported mutations in
BRCA1 result in truncated proteins. Primers
were designed to amplify exon 11, which
encodes 61% of the BRCA1 gene product.
Members of 35 families were analyzed, and all
produced the correct size of PCR(f) product from
the exon. The PCR product was transcribed
and translated in vitro with [35S]methionine and
analyzed by SDS-PAGE and autoradiography.
Six mutations resulting in truncated proteins
were identified. The mutant PCR products were
directly sequenced and were found to be the
result of either insertions or deletions yielding
frameshift mutations and premature stop
codons. Genomic DNA has also been used to
analyze the genes BRCA2 (13), APC (14,15)
and PLEC1 (16) by PTT.
Use of genomic DNA as the source of nucleic
acid for PTT has some drawbacks in that indi-
vidual exons must be analyzed. To analyze the
entire coding sequence of a gene like BRCA1,
24 individual exons would need to be amplified
and analyzed. Besides requiring a large number of amplifications, assuming all the exons are
large enough to translate, analysis of the individual exons could miss truncation mutations
that could result in aberrant exon splicing. In the
same study that amplified exon 11 of the
BRCA1 gene from genomic DNA for PTT analysis, Hogervorst et al. (12) isolated total RNA
from freshly isolated peripheral blood lymphocytes. The sequences corresponding to exons
2–10 were amplified by RT-PCR(f) and analyzed
by PTT. One subject had a mutation in one
allele that resulted, first, in a smaller RT-PCR
product and, second, in a truncated protein by
PTT. The mutation was directly sequenced and
resulted from aberrant splicing of exons 9 and
10. Thus, using RT-PCR and PTT, larger portions
of a gene can be amplified and analyzed, picking up aberrant splicing mutations not identified
by analysis of the exons via amplification of
genomic DNA. In most cases, when RT-PCR is
used as the method to generate targets, the
entire coding region is broken into several
smaller fragments. For example, three amplifications were used to test the entire coding region
of the TSC2 gene by PTT (17). When using multiple targets to span an entire coding region, the
amplimers should overlap so that a mutation at
the 3′-end of one target (that does not cause a
References (continued)
12. Hogervorst, F.B.L. et al. (1995)
Nat. Genet. 10, 208.
13. Lancaster, J.M. et al. (1996) Nat.
Genet. 13, 238.
14. Powell, S.M. et al. (1993) New
Eng. J. Med. 329, 1982.
15. van der Luijt, R. et al. (1994)
Genomics 20, 1.
16. Dang, M. et al. (1998) Lab.
Invest. 78, 195.
17. van Bakel, I. et al. (1997) Hum.
Mol. Genet. 6, 1409.
18. Hogervorst, F.B.L. (1997)
Promega Notes 62, 7.
19. Transcend™ Non-Radioactive
Translation Detection Systems
Technical Bulletin #TB182,
Promega Corporation.
20. Kirchgesser, M. et al. (1998)
Clin. Chem. Lab. Med. 36, 567.
21. Rowan, A.J. and Bodmer, W.F.
(1997) Hum. Mutat. 9, 172.
22. Eccles, D.M. et al. (1996) Am. J.
Hum. Genet. 59, 1193.
23. Wright, J. et al. (1996) Am. J.
Hum. Genet. 59, 839.
24. Bai, M. et al. (1997) J. Clin.
Invest. 99, 1917.
25. Romey, M. et al. (1996) Hum.
Genet. 98, 328.
26. Beaufrère, L. et al. (1997) Exp.
Eye Res. 65, 849.
27. Gardner, R.J. et al. (1995) Am.
J. Hum. Genet. 57, 311.
28. Lo Ten Foe, J.R. et al. (1996)
Nat. Genet. 14, 320.
29. Pegoraro, E. et al. (1998)
Neurology 51, 101.
30. Liu, B. et al. (1994) Cancer Res.
54, 4590.
31. Papadopoulos, N. et al. (1994)
Science 263, 1625.
32. Heim, R.A. et al. (1994) Nat.
Genet. 8, 218.
33. MacCollin, M. et al. (1994) Am.
J. Hum. Genet. 55, 314.
34. Axton, R. et al. (1997) J. Med.
Genet. 34, 279.
35. Maugard, C. et al. (1997) Br. J.
Haematol. 98, 21.
36. Roelfsema, J.H. et al. (1996)
Nephrol. Dial. Transplant.
11(suppl. 6), 5.
Table 1. Genes Analyzed with the Protein Truncation Test a.
Condition
Familial Adenomatous Polyposis
Hereditary Desmoid Disease
Ataxia Telangiectasia
Hereditary Breast and Ovarian Cancer
Familial Hypocalciuric Hypercalcemia
Cystic Fibrosis
Chorioderemia
Duchenne Muscular Dystrophy
Fanconi Anaemia
Congenital Muscular Dystrophy
Hereditary Non-Polyposis Colorectal Cancer
Neurofibromatosis Type 1
Neurofibromatosis Type 2
Aniridia
Paroxysmal Nocturnal Haemoglobinuria
Polycystic Kidney Disease
Epidermolysis Bullosa with Muscular Dystrophy
Dystrophic Epidermolysis Bullosa
Breast Cancers, Gliomas, Melanomas
Rubenstein-Taybi Syndrome
Familial Tuberous Sclerosis
aMore
Gene
APC
APC
ATM
BRCA1
BRCA2
CASR
CFTR
CHM
DMD
FAA
laminin-α2
hMSH2
hMLH1
NF1
NF2
PAX6
PIG-A
PKD1
PLEC1
COL7A1
PTEN/MMAC1
RTS
TSC2
Ref.
14,15
22
23
12
13
24
25
26
7,27
28
29
30
31
32
33
34
35
36,37
16,44–46
43
38,39,40
41
17
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references available in The Protein Truncation Test Bibliography (BL002) and Mutation Detection (BR043) also available on the Internet at www.promega.com
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significant change in molecular weight) will be
detected in another target having the same
codons near the 5′-end.
References (continued)
37. Roelfsema, J.H. et al. (1997)
Am. J. Hum. Genet. 61, 1044.
38. Li, J. et al. (1997) Science 275,
1943.
39. Furnari, F.B. et al. (1997) Proc.
Natl. Acad. Sci. USA 94, 12479.
40. Robertson, G.P. et al. (1998)
Proc. Natl. Acad. Sci. USA 95,
9418.
41. Petrij, F. et al. (1995) Nature 376,
348.
42. Sarkar, G. and Sommer, S.S.
(1989) Science 244, 331.
43. Whittock, N.V. et al. (1999) J.
Invest. Dermatol. 113, 673.
44. Takizawa, Y. et al. (1999) J.
Invest. Dermatol. 112, 109.
45. Kunz, M. et al. (2000) J. Invest.
Dermatol. 114, 376.
46. Rouan F. et al. (1989) J. Invest.
Dermatol. 114, 381.
Detection and Primer Design
The detection method for PTT products must be
considered when designing primers for amplification (18). Typically, [35S]methionine is the
label of choice but other labels such as
[35S]cysteine and [3H]leucine could be used as
well. Thus, the amplified segments should contain one or more of these amino acids. The
reactions are resolved on an SDS-PAGE gel and
either directly dried or fluorographically
enhanced and exposed to X-ray film (9). The
dried gels can also be analyzed by phosphorimaging. When radioactive incorporation is not
an option, non-radioactive techniques are available. Proteins can be tagged with biotin by
inclusion of biotinylated lysine tRNA in the translation reaction (9,19). The biotin moiety is then
detected with a streptavidin-enzyme conjugate
and developed via either a colorimetric or
chemiluminescent reaction (19). For example,
PTT has been applied to the APC gene using
translation with a biotinylated lysine tRNA (20).
Other methods for non-radioactive detection
include the inclusion of an epitope tag in the 5′primer so that the translation products can be
analyzed by Western blotting with an antibody
that binds the epitope (21). When dealing with a
heterozygous condition, both the normal and
mutant targets will be amplified and both the
truncated and full-length protein will be detected, unless the allelle is on the X or Y
chromosome of male subjects, no matter which
detection method is chosen.
PTT offers a quick and easy method for analyzing a protein coding sequence for truncation
mutations. However, the method has some limitations. If the truncated sequence does not
translate well or does not contain the appropriate amino acid for labeling, the mutation could
be overlooked. Also, if the truncation is very
near the 3′-end of the target, truncation could
be missed due to the inability of SDS-PAGE to
resolve such differences. If the mutations are
very near the 5′-end of the coding sequence,
the mutation could be missed as well.
Refinements of PTT detection, such as the
incorporation of an epitope tag into the 5′ PCR
primer (21), could allow detection of these
mutations, since incorporation of a specific
amino acid is not needed for detection. Finally,
incorporation of fluorescence-tagged amino
acids may simplify the detection of proteins by
PTT and can possibly be used for quantitation
of the mutant protein (18).
Table 2. Sequences of Different T7-Modified Oligonucleotide Primers for In Vitro Transcription
and Translationa.
Restriction
Site Sequence
GGATCC
GGATCC
GGATCC
nnnb
T7 Bacteriophage
Sequence
TAATACGACTCACTATAGGG
TAATACGACTCACTATAGGG
TAATACGACTCACTATAGG
TAATACGACTCACTATAGG
Spacer
AG
AG
AACAG
AACAG
Eukaryotic
Translation
Initiation
Sequence
CCACC ATG
CCACC ATG G
CCACC ATG
CCACC ATG G
Ref.
13,42
30,31
7,15
12,28
provided are for only the upstream portion of the 5 ′ primer that is not gene specific. For gene-specific use, the eukaryotic translation
initiation sequence would be followed by 17–20 bases exactly complementary to the sequence of interest.
bn = any nucleotide
aSequences
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